Human papillomaviruses cause ubiquitous infectious of the keratinised epithelia of the skin and of the mucosae. About 120 HPV types have been characterized so far, which differ in prevalence, epidemiology and clinical manifestations (de Villiers et al., 2004). In particular, mucosal types infect the keratinised epithelia of the genital, anal and oro-pharyngeal mucosae (Muñoz et al., 2003; Muñoz and Bosch, 1997; Van Ranst et al, 1992; Chan et al., 1995, D'Souza et al., 2007, Bosh et al., 2008). Mucosal HPVs are most commonly transmitted by sexual contact, and infect sexually active people with a very high prevalence. It is estimated that the lifetime incidence of HPV infection in women is 80% (Bekkers at al., 2004), and the overall prevalence of active infection worldwide varies form 1.4% to 25% (Clifford at al, 2005).
Although the vast majority of infections are benign and self-limiting, a subset of “high risk” HPV types have the potential to cause persistent infection that may progress to malignant transformation and invasive cancer (Muñoz et al., 2003). Cervical cancer is the most common HPV-associated malignancy and it is now clear that HPV is a necessary cause of virtually all cervical cancers (Bosch and Muñoz, 2002; zur Hausen, 2002, Bosch et al., 2002; Muñoz et al., 2003; Walboomers et al., 1999, Smith et al, 2007). HPV associated malignancies are also found in the anal canal (Melbye and Sprogel, 1991; Palefsky et al., 1991), vulva (Buscema et al., 1988), the penis (Gregoire et al., 1995; Iwasawa et al., 1993), oro-pharyngeal mucosae and other head and neck tissues (D'Souza, et al., 2007; Mork et al., 2001; Gillison et al., 2000; Syrjanen, 2005).
Since HPV infection is necessary for the development of virtually all cervical cancers, detection of high risk HPV types is being considered as a screening method for cervical cancer, alongside, or even in substitution of, traditional cytological screening using the Papanicolau methods (pap test), with the promise of improving the sensitivity and cost effectiveness of cervical cancer screening programs (Cuzik et al., 2008; Cuzick et al., 2003; Ronco et al., 2006; Schiffman et al., 2005; Kim et al., 2005; Davies et al., 2006; Mayrand et al., 2006; Cuzik et al., 2006).
Two type-specific HPV vaccines (Gardasil, from Merck-Frosst for types 16, 18, 6 and 11; Cervarix form Glaxo-Smith-Kline for types 16 and 18) have recently been developed and clinical trials have shown that they are extremely effective in preventing both persistent infection with HPV and the dysplastic changes in the cervical epithelium that lead to malignant transformation (Koutsky et al., 2002; Villa et al., 2005; Harper et al., 2004; Harper et al., 2006). However, since vaccines are type-specific it is important to know the distribution of the various HPV types in a population, as well as to have a surveillance system in place to monitor vaccine efficacy and unexpected shifts in the frequency of HPV types not covered by the vaccines.
It is therefore expected that the routine use of type-specific tests for HPV will become more widespread, outside their current use in epidemiological studies for research purposes.
Currently, typing of HPV requires amplification by various PCR methods, followed by detection of specific sequences using either direct sequencing of the PCR products, RFLP methods (many methods have been described in the literature, for example Lungo et al., 1992; Menzo et al., 2008, Nobre et al., 2008; Santiago et al., 2006), Southern blot or dot blot using specific probes (for example Gregoire et al., 1989; Josefsson et al., 1999), reverse line hybridization (Gravitt et al., 1998; Kleter et al., 1999; van der Brule et al., 2002; Melchers et al, 1999), DNA microarray methods (Min et al., 2006; Albrecht et al, 2006; Choi et al., 2003; Huang et al., 2004; Hwang eta la., 2003; Oh et al., 2004; Nuovo et al., 2008), and others (for example Nishiwaki et al., 2008; Dell'Atti, 2007; Gao et al., 2003; Gharizadeh et al., 2007; Han et al., 2006; Lee et al., 2005; Liu et al, 2003; Zhang et al, 2003). In particular, reverse line blot methods have been validated and have been used extensively for epidemiological studies. Two leading commercial genotyping methods, InnoLiPA (van Hamont, 2006) and Roche linear array (Coutlee et al., 2006), are based on the reverse hybridization technology. The Roche Linear Array genotyping kit as been approved by FDA and it is the leading commercial genotyping method. However, these methods are not suitable for high throughput testing and they rely on a subjective visual assessment of band intensity for determining the results.
The xMAP technology developed by Luminex (Austin, Tex., USA) is based on microspheres that can be produced in 100 different “colours” depending on they ratio of two spectrally distinct fluorophores coupled to the microspheres. The different colours can be recognized by flow cytometers and the different type of microspheres can be enumerated and analyzed for the presence of specific bound ligands. This technology has been the basis for a variety of multiplex assays for serology, genotyping and other analytical applications. A description of the Luminex technology and a list of publications can be found at the Luminex web site.
Each type of microsphere can be coupled with a specific ligand, e.g. with DNA probes specific for each type of HPV in this work, and mixed together to form a multiplex assay. The PCR products derived from HPV samples are labelled with biotin and mixed with the beads carrying the probes, so that HPV DNA will hybridize with the cognate probe. The flow cytometer will then sort the different “coloured” microspheres and determine which type carries the fluorescence due to the HPV DNA. The computer software driving the flow cytometer will indicate which beads are fluorescent, thereby identifying the HPV type(s) present in the sample. The advantages of this method is the low cost per assay, the possibility of automation for a high throughput assay, and the flexibility derived from the possibility of adding or removing types of microspheres depending on the need of the assay or on the discovery of new types. Several microsphere-based multiplex assay for HPV genotyping have been published. The method by Wallace et al. (2005) is a multiplex microsphere assay with probes for 45 mucosal HPV. However, formal validation was performed for only a few types and only 20 types were detected from clinical samples, without independent validation of the genotyping result. The method published by Oh et al. (2007) detects 15 HPV types and it has been validated against a 132 clinical samples. A 56 sample comparison with a DNA microarray genotyping method is also shown. The method, by Schmitt et al. (2006), has been carefully validated with HPV plasmids and clinical samples and covers the 22 most common mucosal HPV types. The method by Jiang et al. (2006) describes specific probes for 26 HPV mucosal types. Validation was performed with synthetic oligonucleotides complementary to the probes and with a limited number of clinical samples. A commercial method developed by Qiagen (Hilden, Germany) is able to type 18 HPV high-risk using a proprietary set of primers, followed by detection using a Luminex system. At least one study comparing this Luminex Qiagen test to a reverse line blot hybridization has been published (Seme et al., 2009).
Herein, we report the design of novel HPV type-specific probes and the development of a microsphere multiplex assay that can detect 46 different mucosal types in a single reaction. In addition the unique probe set, compared to the previous method we introduce 2 innovations: i) the use of longer probes (30 mers) to provide for a greater specificity for variants and closely related types; ii) the production of single stranded DNA products by selective digestion of the PCR products with exonuclease, which produces a greater signal to noise ratio, making a washing step unnecessary.